An approximately 1.2-Mb bacterial artificial chromosome contig refines the genetic and physical maps of the lurcher locus on mouse chromosome 6

Abstract

Lurcher (Lc) is a semidominant mouse mutant that displays a characteristic ataxia in the heterozygous state beginning in the third postnatal week. This symptom results from a neurodegenerative event in the cerebellum: There is a catastrophic loss of Purkinje cells in the heterozygote animal between postnatal days 10 and 15. In an effort to identify the genetic lesion borne by Lc mice, we initiated a cloning project based on the position of the Lc mutation on mouse chromosome 6. We have extended our previous analysis of the genomic segment containing the Lc locus by isolating a set of stable and manipulable genomic clones called bacterial artificial chromosomes (BACs) that cover this region of mouse chromosome 6. These clones provided a good substrate for the isolation of markers that were used to refine the physical map of the locus. Furthermore, 20 of these markers were mapped onto our (B6CBACa-AW-J/A-Lc x CAST/Ei)F1 x B6CBACa-AW-J/A backcross, refining the genetic map and identifying two nonrecombinant markers (D6Rck354 and D6Rck355). These two markers, in conjunction with the closest flanking markers, were used to identify a 110-kb genomic segment that contains all four markers and hence contains the Lc locus. This small genomic segment, covered by multiple BACs, sets the stage for the final effort of this project-the identification of transcripts and of the mutation within the Lc locus.

Figure 1

A composite physical map of the Lc locus on mouse chromosome 6. The markers presented here are a selection of markers from Table 1 that illustrate the redundancy of the YAC and BAC contigs. (Top) A diagram of the chromosomal segment under study showing the relative position of the markers on the chromosome. The YAC contig is shown in the center; individual YACs are labeled with their respective names. The BAC contig is found at the bottom; individual BACs are labeled with their abbreviated names. The position of each marker is indicated by a bold vertical line on the chromosome, YACs and BACs; markers that are end clones are indicated with a • on the YAC or BAC from which they were isolated. The BAC contig stretches from D6Rck342 to D6Rck329, covering ∼1.2 Mb. The Lc mutation is contained within the genomic segment defined by markers D6Rck353 and D6Rck357. Two genes were mapped to positions within the BAC contig; a short line describes the general position of these genes. More specifically, Lt1 maps to marker D6Rck329 and Atoh1 to D6Rck368.

Figure 2

Genotyping of the six critical animals with markers in the Lc locus of mouse chromosome 6. These animals—CL103, CL222, CL230, CL340, CL369, and CL391—have recombination events within 1 Mb of the Lc locus; however, CL222 and CL340 have recombination events that map outside the D6Rck342–D6Rck329 genomic segment that is under study. The phenotypic status of each animal is indicated beneath the name of the animal. The markers used in this study are listed in a column to the left of the diagram of mouse chromosome 6. The two chromosomes of each animal are illustrated to the right of the diagram. The polymorphism used to genotype each marker is described in Table 1. Because the position of most of these markers cannot be resolved genetically, their position relative to each other was determined using the physical map (Table 1). The location of the Lc mutation can be inferred by looking for nonrecombinant markers, markers that cosegregate with the mutation in all 504 animals of the Lc/cast. backcross. We would expect all affected animals to have a musculus allele at the nonrecombinant marker [four different strains may have contributions to the genomic segment containing the Lc locus (Zuo et al. 1995): DBA/2J, C3HeB/FeJ, C57BL/6J, and B6CBACa-A^w − J/A]. In addition, all wild-type mice should have the wild-type allele, the CAST/Ei allele, at the nonrecombinant marker. This pattern is seen at two markers: D6Rck354 and D6Rck355. The closest flanking markers D6Rck353 and D6Rck357 thus define the genomic segment containing the Lc locus.

Figure 3

Genetic mapping of the nonrecombinant marker D6Rck354 by SSCP. The first seven lanes contain a panel of control reactions; the origin of the substrate genomic DNA is indicated at the top of each lane. The following seven lanes contain the products of the reactions using the genomic DNA of the critical animals as substrate. These six animals are CL103, CL222, CL230 (two lanes), CL340, CL369, and CL391. The phenotypic status of these animals is described in Fig. 2. Finally, the last 15 lanes contain the products of reactions amplified from the genomic DNA of 15 control animals from the backcross. None of the latter animals has a recombination event within 1 Mb of the Lc locus. Two bands are highlighted with arrows. The higher band (1) is CAST/Ei-specific; the lower band (2) is specific to the inbred strains tested here (DBA/2J, C3HeB/FeJ, C57BL/6J, and B6CBACa-A^w − J/A). Some of the lanes—especially animals CL222, CL230, CL340, and CL391—in this gel appear to have a signal strength that is much weaker than that of the control lanes; however, because other bands in the affected lanes have a proportionally lower signal, the difference among the lanes probably arises from differences in the efficiency of certain PCR reactions. A longer exposure of this gel presents the expected pattern of a nonrecombinant marker.

Figure 4

Restriction map of the Lc locus. Partial digests of BACs 143E21 and 222L15 with either BssHII (B) or SfiI (S) and with both BssHII and SfiI were separated by PFGE. The two nonrecombinant markers, D6Rck354 and D6Rck355, and the two flanking markers, D6Rck353 and D6Rck357, were hybridized to the resulting blots and defined a 110-kb segment that contains all four markers. The positions of both the markers and the restriction sites are noted on a diagram of 143E21; the genomic segment containing the Lc locus is outlined below.